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Search Results: 1 - 10 of 303651 matches for " Leonardo J Richtzenhain "
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ISOLATION OF Rickettsia bellii FROM Amblyomma ovale AND Amblyomma incisum TICKS FROM SOUTHERN BRAZIL
Richard Pacheco,Simone Rosa,Leonardo Richtzenhain,Matias P. J. Szabó
Revista MVZ Córdoba , 2008,
Abstract: Objective. To isolate and characterize rickettsiae from the ticks Amblyomma ovale and Amblyomma incisum collected in the state of S o Paulo. Materials and methods. Adult, free-living A. ovale and A. incisum were collected in an Atlantic rainforest area in the state of S o Paulo, Brazil. Each tick was tested using the hemolymph assay; samples from positive ticks were placed in shell vials in order to isolate rickettsiae and subsequently grown in Vero cells. Amplification of three rickettsial genes (gltA, htrA and ompA) was attempted using polymerase chain reaction (PCR) for each isolate obtained. Amplicons were subsequently sequenced. Results. A total of 388 A. incisum and 50 A. ovale were collected. Only one A. incisum and one A. ovale were hemolymph-test positive. Rickettsiae were successfully isolated from these ticks; however establishment in Vero cell culture was successful only for the isolate from A. ovale. Bacterial contamination in the first cell passage of the A. incisum isolate precluded successful isolation of the organism. PCR products were obtained with the gltA and htrA primers for the two isolates, but no product was obtained with the ompA primers. By BLAST analysis, partial gltA and htrA sequences of isolates from A. ovale and A. incisum were similar to the corresponding sequences of R. bellii. Conclusions. This is the first report of R. bellii infecting A. incisum and the first successful isolation from A. ovale.
Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene
Montassier, Maria de Fátima S.;Brentano, Liana;Montassier, Hélio J.;Richtzenhain, Leonardo J.;
Pesquisa Veterinária Brasileira , 2008, DOI: 10.1590/S0100-736X2008000300011
Abstract: twelve brazilian isolates and one reference vaccine strain of avian infectious bronchitis virus (ibv) were propagated in embryonating chicken eggs. the entire s1 glycoprotein gene of these viruses was analysed by reverse-transcriptase-polymerase chain reaction and restriction fragment length polymorphism (rt-pcr-rflp), using the restriction enzymes haeiii, xcmi and bstyi. the rflp patterns led to the classification of these isolates into five distinct genotypes: a, b, c, d and massachusetts. five of twelve isolates were grouped in massachusetts genotype and the remaining seven viruses were classified into four distinct genotypes: a (2), b (2), c (2) or d (1). such genotyping classification agreed with previous immunological analysis for most of these viruses, highlighting the occurrence of a relevant variability among the ibv strains that are circulating in brazilian commercial poultry flocks.
Brucella spp. isolation from dogs from commercial breeding kennels in S?o Paulo state, Brazil
Keid, Lara B.;Soares, Rodrigo M.;Morais, Zenaide M.;Richtzenhain, Leonardo J.;Vasconcellos, Sílvio A.;
Brazilian Journal of Microbiology , 2004, DOI: 10.1590/S1517-83822004000100027
Abstract: dogs from 12 commercial breeding kennels were submitted to clinical investigation and laboratorial tests for diagnosis of brucella spp. infection. the sampling was carried out between april 2000 and february 2002 and the laboratorial tests employed were agar gel immunediffusion test (agid) and blood culture. from 171 dogs examinated, 39 (22.8%) showed at least one clinical sign compatible with brucellosis, 58 (33.91%) were agid positive and 24 (14.03%) were positive by blood culture. gram negative bacterial cells with a biochemical pattern compatible with that of bacteria belonging to genus brucella were isolated from blood specimens of 24 animals. according to kappa index and mcnemar test, the association between agid and blood culture (k=0.360 with 95% of confidence interval; x2=25.93, p=0.000), between agid and clinical test (k=0.248 with 95% of confidence interval; x2=6.11, p=0.013), and between blood culture and clinical examination (k=0.442 with 95% of confidence interval; x2=6.76, p=0.009) were not statistically significant. qui-square test indicated no association of sex and the results of clinical examination (x2=1.35 and p=0.2447), agid (x2=1.58 and p=0.2086) or bacterial isolation (x2=1.48 and p=0.2230). within 12 kennels, seven had at least one dog positive by blood culture and nine had at least one animal positive by agid. the association of epidemiological data with direct and indirect methods of diagnosis is necessary to perform a definitive diagnosis of brucella infection in dogs, as positive results by agid can be consequence of non-specific reactions and must be confirmed by blood culture. negative results by agid must also be confirmed using direct methods of diagnosis or repeating the serologic test after 30 days, because of the low sensitivity of this test.
ISOLATION OF Rickettsia bellii FROM Amblyomma ovale AND Amblyomma incisum TICKS FROM SOUTHERN BRAZIL
Pacheco,Richard; Rosa,Simone; Richtzenhain,Leonardo; Szabó,Matias P. J.; Labruna,Marcelo B;
Revista MVZ Córdoba , 2008,
Abstract: objective. to isolate and characterize rickettsiae from the ticks amblyomma ovale and amblyomma incisum collected in the state of s?o paulo. materials and methods. adult, free-living a. ovale and a. incisum were collected in an atlantic rainforest area in the state of s?o paulo, brazil. each tick was tested using the hemolymph assay; samples from positive ticks were placed in shell vials in order to isolate rickettsiae and subsequently grown in vero cells. amplification of three rickettsial genes (glta, htra and ompa) was attempted using polymerase chain reaction (pcr) for each isolate obtained. amplicons were subsequently sequenced. results. a total of 388 a. incisum and 50 a. ovale were collected. only one a. incisum and one a. ovale were hemolymph-test positive. rickettsiae were successfully isolated from these ticks; however establishment in vero cell culture was successful only for the isolate from a. ovale. bacterial contamination in the first cell passage of the a. incisum isolate precluded successful isolation of the organism. pcr products were obtained with the glta and htra primers for the two isolates, but no product was obtained with the ompa primers. by blast analysis, partial glta and htra sequences of isolates from a. ovale and a. incisum were similar to the corresponding sequences of r. bellii. conclusions. this is the first report of r. bellii infecting a. incisum and the first successful isolation from a. ovale.
Rickettsia felis infection in cat fleas Ctenocephalides felis felis
Horta, Mauricio C.;Scott, Fabio B.;Correia, Thaís R.;Fernandes, Julio I.;Richtzenhain, Leonardo J.;Labruna, Marcelo B.;
Brazilian Journal of Microbiology , 2010, DOI: 10.1590/S1517-83822010000300035
Abstract: the present study evaluated the rickettsial infection in a laboratory colony of cat fleas, ctenocephalides felis felis (bouche) in brazil. all flea samples (30 eggs, 30 larvae, 30 cocoons, 30 males, and 30 females) tested by polymerase chain reaction (pcr) were shown to contain rickettsial dna. pcr products, corresponding to the rickettsial glta, htra, ompa and ompb gene partial sequences were sequenced and showed to correspond to rickettsia felis, indicating that the flea colony was 100% infected by r. felis. the immunofluorescence assay (ifa) showed the presence of r. felis-reactive antibodies in blood sera of 7 (87.5%) out of 8 cats that were regularly used to feed the flea colony. from 15 humans that used to work with the flea colony in the laboratory, 6 (40.0%) reacted positively to r. felis by ifa. reactive feline and human sera showed low endpoint titers against r. felis, varying from 64 to 256. with the exception of one human serum, all r. felis-reactive sera were also reactive to rickettsia rickettsii and/or rickettsia parkeri antigens at similar titers to r. felis. the single human serum that was reactive solely to r. felis had an endpoint titer of 256, indicating that this person was infected by r. felis.
Rapid detection of bovine coronavirus by a semi-nested RT-PCR
Asano, Karen M.;Souza, Sibele P.;Silva, Sheila O.S.;Richtzenhain, Leonardo J.;Brand?o, Paulo E.;
Pesquisa Veterinária Brasileira , 2009, DOI: 10.1590/S0100-736X2009001100001
Abstract: bovine coronavirus (bcov) is a member of the group 2 of the coronavirus (nidovirales: coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. the present study aimed to develop a semi-nested rt-pcr for the detection of bcov based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. the analytical sensitivity was determined by 10-fold serial dilutions of the bcov kakegawa strain (ha titre: 256) in depc treated ultra-pure water, in fetal bovine serum (fbs) and in a bcov-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of rna in the sample influence the precipitation of pellets by the method of extraction used. when fecal samples was used, a large quantity of total rna serves as carrier of bcov rna, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the pcr. the final semi-nested rt-pcr protocol was applied to 25 fecal samples from adult cows, previously tested by a nested rt-pcr rdrp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). the high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current bcov sequences give basis to a more accurate diagnosis of bcov-caused diseases, as well as to further insights on protocols for the detection of other coronavirus representatives of both animal and public health importance.
Ehrlichia canis em c?es atendidos em hospital veterinário de Botucatu, Estado de S?o Paulo, Brasil
Ueno, Tatiana E. H.;Aguiar, Daniel M.;Pacheco, Richard C.;Richtzenhain, Leonardo J.;Ribeiro, Márcio G.;Paes, Ant?nio C.;Megid, Jane;Labruna, Marcelo B.;
Revista Brasileira de Parasitologia Veterinária , 2009, DOI: 10.4322/rbpv.01803010
Abstract: this study investigated the etiology of canine ehrlichiosis and possible clinical and epidemiological data associated with the infection in 70 dogs suspect of ehrlichiosis attended at the veterinary hospital of the s?o paulo state university in botucatu city during 2001 and 2002. dogs were evaluated by clinical-epidemiological and hematological data and molecular analysis by partial amplification and dna sequencing of the ehrlichial dsb gene. e. canis dna was amplified and sequenced in 28 (40.0%) dogs. dogs younger than 12 months old showed significantly higher infection rates (65.0%; p < 0.05). diarrhea, apathy, and anorexia were the major clinical signs observed in 55.2% (p = 0.05), 47.0% (p > 0.05), and 42.4% (p > 0.05) of the pcr-positive dogs, respectively. twenty-five anemic (<5.5 × 106 rbc.μl-1), and 8 leukopenic (<5.5 × 103 wbc.μl-1) dogs were pcr-positive (p > 0.05). all 28 pcr-positive dogs showed thrombocytopenia (<175 × 103 platelets.μl-1) and revealed statistical significance (p < 0.05). e. canis was the only ehrlichia species found in dogs in the studied region, with higher infection rates in younger dogs, and statisticallyassociated with thrombocytopenia.
Detection of porcine circovirus genotypes 2a and 2b in aborted foetuses from infected swine herds in the State of S?o Paulo, Brazil
Alessnadra M.M.G. Castro, Taís F Cruz, Vanessa R Salgado, Tatiana M Kanashiro, Karen L Ferrari, Jo?o P Araujo, Paulo E Brand?o, Leonardo J Richtzenhain
Acta Veterinaria Scandinavica , 2012, DOI: 10.1186/1751-0147-54-29
Abstract: Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of S?o Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n?=?1) or 2b (n?=?10).The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of S?o Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.
Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5'-UTR region
Cortez, Adriana;Heinemann, Marcos B;Castro, Alessandra Marnie M.G. de;Soares, Rodrigo M;Pinto, Ana Maria V.;Alfieri, Amauri A.;Flores, Eduardo F.;Leite, R?mulo Cerqueira;Richtzenhain, Leonardo J.;
Pesquisa Veterinária Brasileira , 2006, DOI: 10.1590/S0100-736X2006000400005
Abstract: nineteen isolates of bovine viral diarrhea virus (bvdv) from brazil were genetically characterized through partial nucleotide sequencing and analysis of the 5'utr region. the isolates were grouped as bvdv-1 (11/19), bvdv-2 (6/19) or "atypical" pestivirus (2/19). among the bvdv-1, eight isolates were classified as subgenotype bvdv-1a, whereas most (4 out of 6) bvdv-2 belonged to subgenotype 2b. two isolates from aborted fetuses were not classified into any genetic group, being considered atypical bvdvs. genetic diversity among brazilian bvdv isolates may be responsible for vaccination and diag-nostic failure and therefore may influence the control strategies for bvdv infection in the country.
Development of a Microplate Lectin-Capture RT-PCR (MLC-RT-PCR) for the Detection of Avian Infectious Bronchitis Virus  [PDF]
Maria de Fatima S. Montassier, Vanessa Mirabeli T. Piza, Cintia Hiromi Okino, Liana Brentano, Leonardo José Richtzenhain, Helio José Montassier
Advances in Microbiology (AiM) , 2013, DOI: 10.4236/aim.2013.33039
Abstract:

Rapid, sensitive and specific methods are necessary to confirm the diagnosis of outbreaks of avian infectious bronchitis virus (IBV) infection. The amplification of IBV genome by reverse transcription followed by polymerase chain reaction (RT-PCR) has been one of the most used methods for the detection of this virus in clinical samples. To reduce the time and the number of steps in the molecular diagnosis of IBV, we developed a sensitive and rapid detection method based on viral capture by a lectin (Concanavalin ACon A) in the microplate wells, followed by RT-PCR to amplify the S1 gene. The detection limit of IBV was 103 EID50/ml for the amplification of 5’part of the S1 gene, and 104 EID50/ml for the amplification of full S1 gene. This technique was specific for IBV detection, and no amplified products were detected for other avian viral pathogens (bursal infectious disease virus, avian metapneumovirus and Newcastle disease virus). The MLC-RT-PCR was as sensitive as conventional RT-PCR, and virus isolation method for the detection of IBV in tissue samples collected from experimentally infected birds. The MLC-RT-PCR technique demonstrated a great potential for the rapid and specific diagnosis of IBV.

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